重组GATA4腺病毒的构建及心肌细胞感染  

Construction of recombinant adenovirus overexpressing GATA4 and its infection in cardiac myocytes

作　　者：李涛[1] 胡晓青[2] 姜科声[1] 周春燕[2] 

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004 [2]北京大学基础医学院生物化学与分子生物学系,北京100191

出　　处：《浙江师范大学学报（自然科学版）》2011年第2期191-196,共6页Journal of Zhejiang Normal University:Natural Sciences

基　　金：国家自然科学基金资助项目(90919022);浙江师范大学博士科研启动基金资助项目(ZC304009172)

摘　　要：利用AdEasy腺病毒表达系统构建了GATA4基因过表达腺病毒.编码大鼠GATA4基因的目的片段克隆入pAdTrackcmv质粒,pAdTtrackcmv-GATA4穿梭质粒经PmeⅠ线性化后转入含pAdEasy-1的BJ5183菌进行同源重组.pAdEsay-GATA4重组质粒经卡那霉素抗性筛选及PacⅠ酶切鉴定.pAdEsay-GATA4转入293A细胞进行包装,产生具有感染性的重组病毒.Ad-GATA4重组病毒感染HeLa及乳鼠心肌细胞,通过免疫印迹及实时定量聚合酶链反应(Real-time PCR)检测GATA4表达及促心肌肥大效应.Ad-GATA4重组病毒感染乳鼠心肌细胞后,诱导心肌细胞表面积明显增加,ANF表达明显增强.结果表明,Ad-GATA4腺病毒成功感染心肌细胞并诱导了大鼠心肌肥大表型的出现.The AdEasy system was used to construct recombinant adenovirus overexpressing GATA4 gene.The target fragment encoding the full-length GATA4 cDNA was inserted into pAdTrackcmv.The pAdTrackcmv-GATA4 was linearized by digestion with Pme Ⅰ,and subsequently cotransformed into E.coli BJ5183 containing pAdEasy-1.Recombinants of pAdEsay-GATA4 were selected by kanamycin resistance and confirmed by digestion with Pac Ⅰ.The pAdEsay-GATA4 was transfected into 293A package cells to generate recombinant adenoviruses Ad-GATA4.HeLa cells and neonatal rat cardiac myocytes were infected with the recombinant adenovirus Ad-GATA4.Then,GATA4 protein expression and its effects on cardiac hypertrophy were detected by Western blot and Real-time PCR.The results showed that GATA4 was overexpressed in Ad-GATA4 infected neonatal cardiomyocytes.The cell surface area was significantly increased and ANF expression was upregulated.Recombinant Ad-GATA4 virus was successfully constructed.The infection of Ad-GATA4 induced the hypertrophy of neonatal cardiomyocytes.

关 键 词：GATA4 重组腺病毒 心肌细胞 心肌肥大 

分 类 号：Q789[生物学—分子生物学]